NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron-sulfur cluster N2 to quinone

Abstract

The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron-sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron-sulfur cluster N2 to quinone.

Figure 1

Structures of the photoaffinity probe (trifluoromethyl)diazirinyl[S-C³H₂]pyridaben ([³H]TDP) and other NADH-quinone oxidoreductase inhibitors. Pyridaben is an analog of the photoaffinity probe with a t-butyl group instead of the (trifluoromethyl)diazirinyl substituent. Rolliniastatin I (structure not shown) is a stereoisomer of bullatacin.

Figure 2

Photoaffinity labeling with [³H]TDP of specific binding sites at apparent molecular masses of 23 and 30 kDa in ETP. (A) SDS/PAGE analysis with a 13.5% gel establishes labeling of only the 23-kDa region at 8 nM [³H]TDP and of 23- and 30-kDa regions at 80 nM. (B) Labeling of the 23-kDa region (high-affinity site) correlates with inhibition of NADH oxidase activity. Labeling of the 30-kDa region (low-affinity site) does not undergo saturation up to 240 nM [³H]TDP. Conditions as in A but varying the [³H]TDP level. Values for [³H]TDP bound at above saturation levels are less reliable than at lower concentrations because of increasing background.

Figure 3

Identification of the photoaffinity-labeled complex I high-affinity binding site protein (molecular mass 23 kDa) as PSST and the 30-kDa protein as ND1 and of the photoaffinity-labeled bacterial NDH-1 site protein (molecular mass 23 kDa) as NQO6. (A) SDS/PAGE analysis as in Fig. 2A but complex I isolated from ETP by blue native PAGE after labeling. Two of the darkest protein bands at 52 and 56 kDa are from complex V. (B) Immunoprecipitation of labeled complex I involved blue native PAGE followed by treatment with anti-Pd NQO6 antibody for PSST and anti-bovine ND1 antibody for ND1. Immunoprecipitation of labeled bacterial membranes involved direct treatment of P. denitrificans and T. thermophilus preparations with anti-Pd NQO6 antibody and anti-Tth NQO6 antibody, respectively.

Figure 4

Scheme showing proposed molecular architecture of complex I components involved in the terminal steps of electron transfer to quinone. See text for details.